Through a combined biochemical and genetic analysis, the objective of the research is to elucidate those enzyme activities in Drosophila that participate in the repair of alkylated DNA. Cells capable of removing alkylated bases from DNA are thought to rely on multiple DNA repair pathways, among which the removal of a single modified base and the re-insertion of the correct base has been suggested. An activity isolated from human fibroblasts, namely purine base insertase, may have direct bearing on this form of alkylated DNA repair. Alternatively, DNA N-glycosylases may remove the modified alkylated base, producing an apurinic DNA lesion susceptible to either base insertion or apurinic/apyrimidinic (AP) endonuclease activity. As well, distinct DNases may specifically recognize individual modified DNA bases as part of a nucleotide excision repair pathway. Purine base insertase and AP endonuclease will be purified from Drosophila melanogaster embryos. The purine base insertase will be subsequently tested for its ability to liberate modified bases from DNA labeled with alkylating agents. If successful, the liberated, modified base will be identified by chromatographic procedures. Beyond the biochemical characterization of purine base insertase, the presence of other alkylated DNA repair proteins in Drosophila, such as DNases and DNA N-glycosylases, will be investigated with an objective toward purifying novel alkylated DNA repair proteins. Alkylation-sensitive Drosophila mutants will be screened to find a mutant that is deficient in AP endonuclease and purine base insertase activity, with an objective toward learning which enzymes represent the predominate repair activity toward a particular DNA insult.